In immune T cells, antigen recognition drives immunological synapse (IS) formation at the interface between T cells and antigen presenting cells (APC). IS formation is a dynamic process during which T cell receptor (TCR) forms microclusters with several downstream signaling molecules at the periphery of T cell-APC contact zone and then move to the center of T cell-APC contact zone leading to the formation of a metacluster called central supramolecular activation cluster (cSMAC). It has been proposed that TCR microclusters initiate and sustain TCR signaling transduction, whereas cSMAC is the site where TCR signaling stops. IS appears to fine-tune and balance TCR signaling, which is essential for proper T cell activation and differentiation to effector cells to elicit immune responses, without development of autoimmunity. Little is known about the molecular mechanism underlying the organization of functional territories in IS. Cdc42 of the Rho GTPase family has been shown, mostly by overexpression of constitutively active or dominant negative mutant of Cdc42 in clonal cell lines that may introduce non-specific effects, to play an essential role in many aspects of cell activities including actin cytoskeleton. This project is motivated by new data generated with mouse Cdc42-deficient T cells to focus on testing the novel hypothesis that Cdc42 serves as an organizer of the functional territories in IS to regulate TCR signaling to control T cell fate and function. The goal of this proposal is to define the molecular mechanisms of Cdc42 in the regulation of IS structural organization and the functional impact of Cdc42-mediated IS structural organization on TCR signaling transduction that leads to T cell activation and differentiation, under physiological settings. By using a mouse model depleted of Cdc42 from immature T cells complemented by a mouse model depleted of Cdc42 in mature T cells, the research will determine whether Cdc42 regulates TCR signaling cluster segregation in IS (Aim 1) and whether Cdc42-mediated IS structural organization is important for TCR signaling that impacts on T cell activation and differentiation (Aim 2). The research will further determine Cdc42 effector pathways in Cdc42-mediated TCR signaling (Aim 3). Successful execution of the project will advance our knowledge of molecular basis of protective immunity and may provide a new signaling principle of T cell regulation that is relevant to diseases such as inflammation and immune deficiency.